The mechanosensitive hair cells of the inner ear consist of a precise array of actin-based stereocilia that insert as rootlets into a dense actin-based meshwork known as the cuticular plate Numerous studies have demonstrated that the proteins that shape the stereocilia are required for normal hearing and balance. On the other hand, little is known about the role the proteins that shape the cuticular plate play in hearing and balance. Indeed, the function that the cuticular plate itself endows to hair bundle development and maintenance remains elusive. Within the cuticular plate, ultrastructural analyses from Tilney?s group in the 1980s revealed numerous types of actin- associated proteins, which presumably control the morphology and the integrity of this unique subcellular structure. Supervillin, a 205 kD actin-crosslinking protein from the villin/gelsolin superfamily, regulates actin dynamics by interacting with non-muscle myosin II. Mutations in MYH9 gene that encodes myosin II are linked to the human hearing loss DFNA17. We showed using RNA in situ hybridization that two isoforms of supervillin; svila and svilc are robustly expressed in zebrafish hair cells of the inner ear. We also demonstrated using our novel Svila rabbit polyclonal antiserum that Svila localizes to the cuticular plate of hair cells in the inner ear of zebrafish. In mice, we showed that Svil is also expressed in the hair cells of the inner ear. In agreement, in mice, in the vestibular type I and type II hair cells, Svil localizes to the cuticular plate at both postnatal day 1 and adult stages. In mice cochlea, Svil localizes to the cuticular plate and near the apical junctional complexes of supporting cells, that surround hair cells, including the head plates of Deiters? and outer pillar cells and the apicolateral margins of inner phalangeal cells. Here, we identify supervillin role in the development and function of the mechanosensory hair cells is to bundle actin filament networks to shape the cuticular plate. We are utilizing genetic and molecular biological techniques along with physiological experiments to test this hypothesized role in a zebrafish model. In this grant proposal we further study the role of supervillin in hair bundle formation and function.